Protein synthesis required for long-term memory is induced by pkc activation on days preceding associative learning

ABSTRACT

The present invention provides methods of contacting a protein kinase C (PKC) activator with a PKC activator in a manner sufficient to stimulate the synthesis of proteins sufficient to consolidate long-term memory. The present invention also provides methods of contacting a protein kinase C (PKC) activator with a PKC activator in a manner sufficient to downregulate PKC.

PRIORITY OF INVENTION

This application is a continuation of U.S. application Ser. No.11,494,636, filed Jul. 28, 2006, which claims priority to U.S.Provisional Application No. 60/703,501 filed Jul. 29, 2005 and U.S.Provisional Application No. 60/728,753 filed on Oct. 21, 2005.

FIELD OF THE INVENTION

The present invention relates to methods of upregulating anddownregulating protein kinase C that are useful for enhancing memory andthe treatment of cell proliferative disorders.

BACKGROUND OF THE INVENTION

Various disorders and diseases exist which affect cognition. Cognitioncan be generally described as including at least three differentcomponents: attention, learning, and memory. Each of these componentsand their respective levels affect the overall level of a subject'scognitive ability. For instance, while Alzheimer's Disease patientssuffer from a loss of overall cognition and thus deterioration of eachof these characteristics, it is the loss of memory that is most oftenassociated with the disease. In other diseases patients suffer fromcognitive impairment that is more predominately associated withdifferent characteristics of cognition. For instance, Attention DeficitHyperactivity Disorder (ADHD), focuses on the individual's ability tomaintain an attentive state. Other conditions include general dementiasassociated with other neurological diseases, aging, and treatment ofconditions that can cause deleterious effects on mental capacity, suchas cancer treatments, stroke/ischemia, and mental retardation.

The requirement of protein synthesis for long-term memory has beendemonstrated over several decades for a variety of memory paradigms.Agranoff et al. (1967) Science 158: 1600-1601; Bergold et al. (1990)Proc. Natl. Acad. Sci. 87:3788-3791; Cavallaro et al. (2002) Proc. Natl.Acad. Sci. 99: 13279-16284; Crow et al. (1990) Proc. Natl. Acad. Sci.87: 4490-4494; Crow et al. (1999) J. Neurophysiol. 82: 495-500; Epsteinet al. (2003) Neurobiol. Learn. Mem. 79: 127-131; Ezzeddine et al.(2003) J. Neurosci. 23: 9585-9594; Farley et al. (1991) Proc. Natl.Acad. Sci. 88: 2016-2020; Flexner et al. (1996) Proc. Natl. Acad. Sci.55: 369-374; Hyden et al. (1970) Proc. Natl. Acad. Sci. 65: 898-904;Nelson et al. (1990) Proc. Natl. Acad. Sci. 87: 269-273; Quattrone etal. (2001) Proc. Natl. Acad. Sci. 98: 11668-11673; Zhao et al. (1999) J.Biol. Chem. 274: 34893-34902; Zhao et al. (2000) FASEB J. 14: 290-300.Flexner originally showed that drug-induced inhibition of proteinsynthesis (e.g., with 5-propyluracil or anisomycin) blocked long-termmemory when this inhibition occurred during a critical time intervalfollowing the training paradigm. Flexner et al. (1996) Proc. Natl. Acad.Sci. 55: 369-374. If protein synthesis was inhibited before thiscritical time window or at any time after this window, there was noeffect on long-term memory. The identity of the proteins essential formemory consolidation, the mechanisms of their regulation, and their rolein the consolidation of long-term memory has remained a mystery.

In many species the formation of long-term associative memory has alsobeen shown to depend on translocation, and thus activation, of proteinkinase C (PKC) isozymes to neuronal membranes. Initially, these PKCisozymes, when activated by a combination of calcium and co-factors,such as diacylglycerol, achieve a stable association with the inneraspect of the external neuronal membrane and membranes of internalorganelle, such as the endoplasmic reticulum. PKC activation has beenshown to occur in single identified Type B cells of the molluskHermissenda (McPhie et al. (1993) J. Neurochem. 60: 646-651), a varietyof mammalian associative learning protocols, including rabbitnictitating membrane conditioning (Bank et al. (1988) Proc. Natl. Acad.Sci. 85: 1988-1992; Olds et al. (1989) Science 245: 866-869), ratspatial maze learning (Olds et al. (1990) J. Neurosci. 10: 3707-3713),and rat olfactory discrimination learning, upon Pavlovian conditioning.Furthermore, calexcitin (Nelson et al. (1990) Science 247: 1479-1483), ahigh-affinity substrate of the alpha isozyme of PKC increased in amountand phosphorylation (Kuzirian et al. (2001) J. Neurocytol. 30: 993-1008)within single identified Type B cells in aPavlovian-conditioning-dependent manner.

There is increasing evidence that the individual PKC isozymes playdifferent, sometimes opposing, roles in biological processes, providingtwo directions for pharmacological exploitation. One is the design ofspecific (preferably, isozyme specific) inhibitors of PKC. This approachis complicated by the fact that the catalytic domain is not the domainprimarily responsible for the isotype specificity of PKC. The otherapproach is to develop isozyme-selective, regulatory site-directed PKCactivators. These may provide a way to override the effect of othersignal transduction pathways with opposite biological effects.Alternatively, by inducing down-regulation of PKC after acuteactivation, PKC activators may cause long term antagonism.

Following associative memory protocols, increased PKC association withthe membrane fractions in specific brain regions can persist for manydays (Olds et al. (1989) Science 245: 866-869). Consistent with thesefindings, administration of the potent PKC activator bryostatin,enhanced rats spatial maze learning (Sun et al. (2005) Eur. J.Pharmacol. 512: 45-51). Furthermore, clinical trials with the PKCactivator, bryostatin, suggested (Marshall et al. (2002) Cancer Biology& Therapy 1: 409-416) that PKC activation effects might be enhanced byan intermittent schedule of drug delivery. One PKC activator,bryostatin, a macrolide lactone, activates PKC in sub-nanomolarconcentrations (Talk et al. (1999) Neurobiol. Learn. Mem. 72: 95-117).Like phorbol esters and the endogenous activator DAG, bryostatin bindsto the C1 domain within PKC and causes its translocation to membranes,which is then followed by downregulation.

The non-tumorigenic PKC activator, bryostatin, has undergone extensivetesting in humans for the treatment of cancer in doses (25 μg/m²-120μg/m²) known to cause initial PKC activation followed by prolongeddownregulation (Prevostel et al. (2000) Journal of Cell Science 113:2575-2584; Lu et al. (1998) Mol. Biol. Cell 18: 839-845; Leontieva etal. (2004) J. Biol. Chem. 279:5788-5801). Bryostatin activation of PKChas also recently been shown to activate the alpha-secretase thatcleaves the amyloid precursor protein (APP) to generate the non-toxicfragments soluble precursor protein (sAPP) from human fibroblasts(Etcheberrigaray et al. (2004) Proc. Natl. Acad. Sci. 101: 11141-11146).Bryostatin also enhances learning and memory retention of the ratspatial maze task (Sun et al. (2005) Eur. J. Pharmacol. 512: 45-51),learning of the rabbit nictitating membrane paradigm (Schreurs andAlkon, unpublished), and in a preliminary report, Hermissendaconditioning (Scioletti et al. (2004) Biol. Bull. 207: 159).Accordingly, optimal activation of PKC is important for many molecularmechanisms that effect cognition in normal and diseased states.

Because the upregulation of PKC is difficult to achieve withoutdownregulation, and vice versa, methods of upregulation of PKC whileminimizing downregulation are needed to enhance the cognitive benefitsobserved associated with PKC activation. The methods and compositions ofthe present invention fulfill these needs and will greatly improve theclinical treatment for Alzheimer's disease and other neurodegenerativediseases, as well as, provide for improved cognitive enhancementprophylactically. The methods and compositions also provide treatmentand/or enhancement of the cognitive state through the modulation ofα-secretase.

SUMMARY OF THE INVENTION

This invention relates to a method of contacting a PKC activator withprotein kinase C in a manner sufficient to stimulate the synthesis ofproteins sufficient to consolidate long term memory.

In one embodiment, the PKC activator is a macrocyclic lactone. In oneembodiment, the PKC activator is a benzolactam. In one embodiment, thePKC activator is a pyrrolidinone. In a preferred embodiment, themacrocyclic lactone is bryostatin. In a more preferred embodiment, thebryostatin is bryostatin-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11,-12, -13, -14, -15, -16, -17, or -18. In the most preferred embodiment,the bryostatin is bryostatin-1.

In one embodiment, the macrocyclic lactone is neristatin. In a preferredembodiment, the neristatin is neristatin-1.

In one embodiment, the contact activates PKC. In one embodiment, thecontact increases the amount of PKC. In one embodiment, the contactincreases the synthesis of PKC. In one embodiment, the contact increasesthe amount of calexcitin. In one embodiment, the contact does not resultin substantial subsequent deregulation of PKC.

In one embodiment, the contact is repeated. In another embodiment, thecontact is repeated at regular intervals. In another embodiment, theinterval is between one week to one month, one day and one week, or lessthan one hour and 24 hours. In another embodiment, the interval isbetween one week and one month. In another embodiment, the interval isbetween one day and one week. In another embodiment, the interval isbetween less than one hour and 24 hours.

In one embodiment, the contact is maintained for a fixed duration. Inanother embodiment, the fixed duration is less than 24 hours. In anotherembodiment, the fixed duration is less than 12 hours. In anotherembodiment, the fixed duration is less than 6 hours. In anotherembodiment, the fixed duration is less than 6 hours. In anotherembodiment, the fixed duration is less than 4 hours. In anotherembodiment, the fixed duration is less than 2 hours. In a preferredembodiment, the fixed duration is between about 1 and 12 hours. In amore preferred embodiment, the fixed duration is between about 2 and 6hours. In the most preferred embodiment, the fixed duration is about 4hours.

In one embodiment, the contact is repeated for a period greater than oneday. In another embodiment, the contact is repeated for a period betweenone day and one month. In another embodiment, the contact is repeatedfor a period between one day and one week. In another embodiment, thecontact is repeated for a period between one week and one month. Inanother embodiment, the contact is repeated for a period between onemonth and six months. In another embodiment, the contact is repeated fora period of one month. In another embodiment, the contact is repeatedfor a period greater than one month.

The invention relates to a method of contacting a PKC activator withprotein kinase C in a manner sufficient to downregulate PKC.

In one embodiment, the PKC activator is a macrocyclic lactone. In oneembodiment, the PKC activator is a benzolactam. In one embodiment, thePKC activator is a pyrrolidinone. In a preferred embodiment, themacrocyclic lactone is bryostatin. In a more preferred embodiment, thebryostatin is bryostatin-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11,-12, -13, -14, -15, -16, -17, or -18. In the most preferred embodiment,the bryostatin is bryostatin-1.

In one embodiment, the macrocyclic lactone is neristatin. In a preferredembodiment, the neristatin is neristatin-1.

In one embodiment, the contact does not stimulate the synthesis of PKC.In another embodiment, the contact does not substantially stimulate thesynthesis of PKC. In another embodiment, the contact decreases theamount of PKC. In another embodiment, the contact substantiallydecreases the amount of PKC. In another embodiment, the contact does notstimulate the synthesis of calexcitin.

In one embodiment, the contact is for a sustained period. In oneembodiment, the sustained period if between less than one hour and 24hours. In another embodiment, the sustained period is between one dayand one week. In another embodiment, the sustained period is between oneweek and one month. In another embodiment, the sustained period isbetween less than one hour and 12 hours. In another embodiment, thesustained period is between less than one hour and 8 hours. In anotherembodiment, the sustained period is between less than one hour and 4hours. In a preferred embodiment, the sustained period is about 4 hours.

In one embodiment, the contact produces sustained downregulation of PKC.

This invention relates to a method of contacting a PKC activator withprotein kinase C in a manner sufficient to stimulate the synthesis ofproteins sufficient to consolidate long term memory, further comprisingthe step of inhibiting degradation of PKC.

In one embodiment, the degradation is through ubiquitination. In anotherembodiment, the degradation is inhibited by lactacysteine. In anotherembodiment, the PKC is human.

This invention relates to a method of contacting a PKC activator withprotein kinase C in a manner sufficient to stimulate the synthesis ofproteins sufficient to consolidate long term memory, wherein the PKCactivator is provided in the form of a pharmaceutical compositioncomprising the PKC activator and a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical composition further comprises aPKC inhibitor. In another embodiment, the PKC inhibitor is a compoundthat inhibits PKC in peripheral tissues. As used herein, “peripheraltissues” means tissues other than brain. In another embodiment, the PKCinhibitor is a compound that preferentially inhibits PKC in peripheraltissues. In another embodiment, the PKC inhibit is a compound thatreduces myalgia associated with the administration of a PKC activator tosubjects in need thereof. In another embodiment, the PKC inhibitor is acompound that reduces myalgia produced in a subject treated with a PKCactivator. In another embodiment, the PKC inhibitor is a compound thatincreases the tolerable dose of a PKC activator. Specifically, PKCinhibitors include, for example, but are not limited to vitamin E,vitamin E analogs, and salts thereof; calphostin C; thiazolidinediones;ruboxistaurin, and combinations thereof. As used herein, “vitamin E”means α-tocopherol (5, 7, 8-trimethyltocol); β-tocopherol (5,8-dimethyltocol; δ-tocopherol (8-methyltocal); and γ-tocopherol(7,8-dimethyltocol), salts and analogs thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawings executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 depicts the effects of bryostatin on long term memoryacquisition, and shows that animals trained sub-optimally, but treatedwith bryostatin, all demonstrate acquisitioned long-term memory. Animalswere trained with sub-optimal regimes of 4- and 6-paired CS/US trainingevents (TEs) with bryostatin (0.25 ng/ml) added during dark adaptation(10 min) prior to training and remaining for 4 hours, or without Bryo(NSW controls); 9-paired TEs and NSW served as the positive controls.Animals were tested with the CS alone at 4 h, then at 24-h intervals.Animals trained sub-optimally but treated with bryostatin alldemonstrated long-term retention (n=8-16 animals/condition/experiment;ANOVA, p<0.01).

FIG. 2 depicts the effects of bryostatin on long-term memoryacquisition, and shows that randomized presentations of light androtation, either with or without bryostatin, produced no conditionedresponse. Long-term memory (LTM) retention effects of Bryostatin undercontrol and antagonist experimental regimes. Without Bryostatin (NSW),random, and paired CS/US training events (TEs) did not generate LTM orelicit a CR when tested at 4 h. Bryostatin (0.25 ng/ml in NSW) appliedbefore 6-TE conditioning (during 10 min dark adaptation) and for 4 hoursthereafter produced a positive CR (foot contraction; negative change inlength), thus indicating LTM was established. The antagonist, Ro-32 whenapplied pre-training (during dark adaptation), blocked the effects of 6TE plus bryostatin, i.e. animals lengthened (positive length change)with normal photaxis (n=4-8 animals/condition/experiment; ANOVAdifferences, p<0.01).

FIG. 3 depicts the effects of bryostatin on long-term memoryacquisition, and shows that animals exposed to bryostatin for four hourson two successive days, followed by two training events (TE) on a thirdsubsequent day, demonstrated acquisition of at least six days oflong-term memory. Two successive days of 4-h bryostatin exposure (0.25ng/ml) of animals coupled with 2-paired CS/US training events producedat least 6 days of long-term retention demonstrated by the CR (bodylength contraction) when tested with the CS alone (n=16animals/condition; ANOVA, p<0.01).

FIG. 4 depicts the effects of bryostatin on long term memoryacquisition, and shows that animals exposed to bryostatin for four hourson three successive days, followed by two TE on a fourth subsequent day,demonstrated acquisition of at least ninety-six hours of long-termmemory. Animals given three successive days of 4-h bryostatin exposure(0.25 ng/ml) followed one day later by 2-TEs, demonstrated long-termretention (LTR) measured over 96 h post-training. Non-exposed animals(same as in FIG. 3) did not demonstrate any behavioral modification (noCR to CS testing). Anisomycin (ANI) (1 μg/ml) administered immediatelyand remaining for four hours post-training to animals receive thethree-day bryostatin treatments did not prevent long-term retention.Thus the requirement for protein synthesis necessary to generate LTRthat is usually blocked by ANI when added post-training was obviated bythe three-day bryostatin treatment (n=16 animals/condition; ANOVA,p<0.01).

FIG. 5 depicts the effects of bryostatin on long term memoryacquisition, and shows that exposure to bryostatin for 8 to 20 hoursfollowed by two TE was not sufficient to acquire memory equivalent tothat achieved after a 4-hour exposure to bryostatin. Effects of 20 hrBryostatin (0.25 ng/ml) exposure on training. With the sub-optimal2-paired TE conditioning regime, retention was gone in 48 hours.Retention of 4-paired TE conditioning with 20 h pre-exposure tobryostatin persisted (n=8 animals/condition; ANOVA at 48-h, p<0.01).

FIG. 6 depicts the effects of bryostatin on long term memoryacquisition, and shows that exposure to more than 1.0 ng/ml ofbryostatin inhibits acquisition of long-term memory. Retentiondose-response curves for 4- and 9-paired CS/US training events.Bryostatin concentrations <0.50 ng/ml augment acquisition and memoryretention with sub-optimal (4 TE) training conditions. Thoseconcentrations had no demonstrable effects on retention performance with9-paired TEs. However, with all training conditions tested,concentration ^(≧)1.0 ng/ml inhibited acquisition and behavioralretention (n=16 animals/condition), presumably via PKC down regulation.

FIG. 7 depicts the effects of bryostatin and anisomycin on long-termmemory acquisition, and shows that a single 4-hour exposure tobryostatin together with 2 TE produced long-term memory lasting hoursthat was entirely eliminated when anisomycin was present duringbryostatin exposure. Bryostatin and anisomycin effects on behavioralacquisition and retention. Animals received 2-paired training events(TEs) and then tested for retention after 4 h. Bryostatin (0.25 ng/ml)applied in NSW to animals during the 10-min pre-training dark adaptationperiod and 4 h thereafter demonstrated retention of the behavioralconditioning (foot contraction (CR) and shortening in body length). NSWcontrol animals and those treated with bryostatin pre-training followedby anisomycin (1.0 μg/ml) immediately post-training showed no CR withthe foot lengthening in normal positive phototaxis (n=12animals/condition/experiment, two-way ANOVA statistics, p<0.01).

FIG. 8 depicts the effects of bryostatin and lactacysteine, and showsthat lactacysteine transformed the short-term memory produced by thesingle bryostatin exposure (followed by 2 TE) to long-term memorylasting many days. Behavioral effects of bryostatin and lactacystin.Animals were incubated simultaneously for 4 h with bryostatin (0.25ng/ml) and lactacystin (10 μM), and then 24 hrs later were conditionedwith 2-paired CS/US training events (TEs). Animals were subsequentlytested with CS alone at 4 h post-training and then at 24-h intervals.Retention of the conditioned behavior was persistent with the combinedbryostatin/lactacystin treatment; behavioral retention was lost bybryostatin-only-treated animals after 24 h. Lactacystin-only treatedanimals showed no acquisition or retention of behavioral training (datanot graphed). (n=28 animals, combined byrostatin/lactacystin; n=20,bryostatin alone, n=16, lactacystin alone).

FIG. 9 depicts the effects of PKC activation on calexcitin. FIGS. (A, B)depict representative tissue sections from Hermissenda eyes that wereimmunolabeled with the calexcitin polyclonal antibody, 25U2. Positiveclaexcitin immunostaining occurred in B-cell photoreceptors (*B-Cell) ofanimals that experienced paired CS/USC associative conditioning with orwithout prior administration of bryostatin (B). Random presentations ofthe two stimuli (training events, TEs) did not produce behavioralmodifications nor a rise in calexcitin above normal background levels(A); basement membrane and lens staining are artifact associated withusing vertebrate polyclonal antibodies. Differences in stainingintensities were measure using Image-J software and recorded asgray-scale intensities (0-256; B-cell cytoplasm minus tissuebackground).

FIG. (C) displays intensity measures for Hermissenda conditioned with9-random TEs (left bar) and animals treated with two exposures onsuccessive days to the PKC antagonist, bryostatin (0.25 ng/ml), and thenassociatively conditioned with 2-paired TEs. Activation of PKC from twoexposures of bryostatin coupled with 2 TEs significantly increasedcalexcitin to levels associated with 9-paired TEs and consolidated(long-term) memory (n=4-8 animals/condition/replicate; t-testcomparison, p<0.01).

Calexcitin immunostaining is sufficiently sensitive to resolve boutonswithin synaptic of photic-vestibular neurites (D). Arrows indicatearborization field between an interneuron (a), axon from a contralateralneuron (b), and terminal boutons of neurites from a putativephotoreceptor (c). Scale bars=10 μm; CPG, cerebropleural ganglion.

FIG. 10 a depicts the effect of bryostatin and training events oncalexcitin immunostaining. The figure shows calexcitin increased withinType B cells with the number of training events. Immuno-intensitymeasurements (as grey-scale intensity; 0-256) of calexicitin (CE)antibody labeling as a function of bryostatin and training regime.Randon traiming (4-TEs) without bryostatin yielded slightly higherintensity measures than background. Bryostatin administration increasedthe calexcitin levels fro both training paradigms. With random training,when there was occasional overlap (pairing) of the CS and US, as was thecase here, it is not unexpected that some rise in CE might occur(increase of 2.0). However, calexcitin levels increased greater than4.3×with paired training (mean±SE, N=5 animals/treatment. 4 RTE=randomcontrol, 4 trials with random light and rotation; 6 PTE=paired trials, 6trials with paired light and rotation. (6 PTE-0 Bry vs. 6 PTE-0.25 Bry;p<0.001; 4 RTE-0.25 Bry vs. 6 PTE-0.25 Bry; p<0.001 (t-test).

FIG. 10 b depicts the effect of bryostatin alone calexcitin, as shown byimmunostaining. Bryostatin alone (without associative conditioning)administered for 4-hr over each of 1, 2, and 3 days progressivelyincreased the levels of calexcitin in the B-photoreceptors ofHermissenda when measured 24 hours after each of the periods ofbryostatin exposures. The calexcitin level after 3 bryostatin exposuresfollowed by just 2-paired training events (paired light and orbitalshaking) raised that level even higher with a significant concomitantlength in the number of retention days for the associativeconditioning-induced behavioral modification (n=16 animals/condition:ANOVA, p<0.01).

FIG. 11 a depicts the effect of 4-hour bryostatin exposure, on twoconsecutive days, followed 24 hours later by two training events, on theintensity of calexcitin. The figure shows that exposure to 4 hours ofbryostatin on two consecutive days followed 24 hours later by 2 TEs arerequired to raise calexcitin levels to the amount associated withconsolidated long-term memory. Exposure to 4-hr of bryostatin on twoconsecutive days followed 24 hours later by 2-training events (2 TE) arerequired to raise calexcitin levels to the amount associated withconsolidated long-term memory, Typically, 2-TEs with two bryostatinexposures produces retention lasting more than one week (n=16animals/condition; t-test, p<0.01).

FIG. 11 b depicts the effect of adding anisomycin after bryostatinexposure on calexcitin. The figure shows that anisomycin following 2 TEplus 3 days of 4 hour bryostatin exposures did not reduce the calexcitinimmunostaining. Priming with 4-hr exposures to bryostatin over 3consecutive days will induce calexcitin levels required for consolidatedmemory. Anisomycin added immediately after the 2-paired training eventsdid not reduce this calexcitin level and consolidated memory persistsfor many days (N=8 animals/condition; t-test, p>0.05, ns).

FIG. 12 depicts the effects of repeated 4-hour bryostatin exposure onPKC activity, as measured by histone phosphorylation in the cytosolicfraction. The figure shows bryostatin exposure on two successiveproduces PKC activity significantly above control or baseline levels.PKC activity in Hermissenda nervous systems (cytosol) after bryostatin.Intact Hermissenda were exposed for 4 hour intervals to bryostatin (0.28nM) on successive days under conditions described (“BehavioralPharmacology”). Histone phosphorylation (See “Methods”) in isolatedcircumesophageal nervous systems was then measured in the cytosolfraction. PKC activity measured both 10 minutes and 24 hours after thesecond of two bryostatin exposures was significantly increased overbaseline levels (N=6, for each measurement).

FIG. 13 depicts the effects of repeated 4-hour bryostatin exposure onPKC activity, as measured by histone phosphorylation in the membranefraction. The figure shows bryostatin exposure on two successiveproduces PKC activity significantly above control or baseline levels.PKC activity in Hermissenda nervous systems (membrane) after bryostatin.As in FIG. 12, histone phosphorylation was measured in the membranefraction. PKC activity measured 24 hours after the second of twobryostatin exposures was significantly increased over baseline (N=6) foreach measurement.

FIG. 14 depicts the effects of anisomycin on PKC activity. The figureshows that the presence of anisomycin during each of three successivedays of bryostatin exposure reduced PKC activity in both cytosolic andmembrane fractions. Anisomycin reduces PKC activity in Hermissenda. Asin FIG. 12, 13 but with anisomycin (1.0 ng/ml) added together with eachbryostatin (0.25 ng/ml) exposure. Note that the anisomycin markedlyreduced the PKC activity in both the cytosolic and membrane fractionsfrom the Hermissenda circumesophagel nervous systems after exposure tobryostatin on three successive days (N=3, for each measurement, p<0.01).

FIG. 15 depicts the effects of bryostatin on membrane-bound PKC inhippocampal neurons. The figure shows that exposure of culturedhippocampal neurons to a single activating dose of bryostatin (0.28 nM)for 30 minutes produced a brief translocation of PKC from the cytosol tothe particulate fraction (approx 60%) followed by a prolongeddownregulation. A second exposure of up to four hours after the firstexposure significantly attenuates the down regulation found four hoursafter a single bryostatin exposure. Effect of bryostatin onmembrane-bound PKC activity in hippocampal cultured IGF/IR cellsafter 1) a single 30 min exposure; or 2) two 30 min exposures separatedby intervals of 30 min to 8 hr. A second exposure up to 4 hr after thefirst exposure significantly attenuates the downregulation found 4 hrafter a single bryostatin exposure (N=6 for each measurement, *p<0.05,**p<0.01).

FIG. 16 depicts the effects of repeated bryostatin exposure on PKCactivity. The figure shows that a second exposure after a 2- to 4-hourdelay eliminated the significant downregulation that a single 30-minutebryostatin exposure produced, and that if the second exposure wasdelayed until 4 hours after the first, activity was increased abovebaseline, to a degree that was significantly greater compared with asecond exposure delivered after 2 hours or less. Effect of bryostatin oncytosolic PKC activity in hippocampal cultured IGF/IR cells after 1) asingle 30 min exposure, or 2) two 30 min exposures separated byintervals of 30 min to 8 hr. PKC activity was not altered in the cytosolthe first 4 hours after bryostatin exposure. By contrast, a secondexposure to bryostatin within 2 hr of the first induced a significantreduction of PKC activity. However, if the second exposure was delayeduntil 4 hours after the first, activity was increased above baseline,and was significantly greater than activity measure in response to asecond exposure delivered after 1 or 2 hours (N=6 for each measurement,*p<0.05, **p<0.01).

FIG. 17 depicts the effects of bryostatin on protein synthesis. RatIGF-IR cells were incubated for 30 minutes with 0.28 nM bryostatin forincubation times ranging from 1 to 79 hours. [³⁵S]Methionine (9.1 μCi)was then added to the medium followed by analysis of radiolabel. Asingle 30-minute exposure to 0.28 nM bryostatin increased overallprotein synthesis, as measured by the incorporation of [³⁵S]Methioninein the last half hour before collecting the neurons, by 20% within 24hours, increasing to 60% by 79 hours after bryostatin exposure, butincreasing significantly less in the presence of the PKC inhibitorRo-32-0432.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

As used herein, “upregulating” or “upregulation” means increasing theamount or activity of an agent, such as PKC protein or transcript,relative to a baseline state, through any mechanism including, but notlimited to increased transcription, translation and/or increasedstability of the transcript or protein product.

As used herein, “down regulating” or “ down regulation” means decreasingthe amount or activity of an agent, such as PKC protein or transcript,relative to a baseline state, through any mechanism including, but notlimited to decreased transcription, translation and/or decreasedstability of the transcript or protein product.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition, compound, or solvent with which an activeingredient may be combined and which, following the combination, can beused to administer the active ingredient to a subject. As used herein,“pharmaceutically acceptable carrier” includes, but is not limited to,one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; preservatives;physiologically degradable compositions such as gelatin; aqueousvehicles and solvents; oily vehicles and solvents; suspending agents;dispersing or wetting agents; emulsifying agents, demulcents; buffers;salts; thickening agents; fillers; antioxidants; stabilizing agents; andpharmaceutically acceptable polymeric or hydrophobic materials and otheringredients known in the art and described, for example in Genaro, ed.(1985) Remington's Pharmaceutical Sciences Mack Publishing Co., Easton,Pa., which is incorporated herein by reference.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, and other mammals.

2. Alzheimer's Disease

Alzheimer's disease is associated with extensive loss of specificneuronal subpopulations in the brain with memory loss being the mostuniversal symptom. (Katzman (1986) New England Journal of Medicine 314:964). Alzheimer's disease is well characterized with regard toneuropathological changes. However, abnormalities have been reported inperipheral tissue supporting the possibility that Alzheimer's disease isa systematic disorder with pathology of the central nervous system beingthe most prominent. (Connolly (1998) Review, TiPS Col. 19: 171-77). Fora discussion of Alzheimer's disease links to a genetic origin andchromosomes 1, 14, and 21 see St. George-Hyslop et al. (1987) Science235: 885; Tanzi et al. Review, Neurobiology of Disease 3:159-168; Hardy(1996) Acta Neuro! Scand: Supplement 165: 13-17.

Individuals with Alzheimer's disease are characterized by progressivememory impairments, loss of language and visuospatial skills andbehavior deficits (McKhann et al. (1986) Neurology 34: 939-944). Thecognitive impairment of individuals with Alzheimer's disease is theresult of degeneration of neuronal cells located in the cerebral cortex,hippocampus, basal forebrain and other brain regions. Histologicanalyzes of Alzheimer's disease brains obtained at autopsy demonstratedthe presence of neurofibrillary tangles (NFT) in perikarya and axons ofdegenerating neurons, extracellular neuritic (senile) plaques, andamyloid plaques inside and around some blood vessels of affected brainregions. Neurofibrillary tangles are abnormal filamentous structurescontaining fibers (about 10 nm in diameter) that are paired in a helicalfashion, therefore also called paired helical filaments. Neuriticplaques are located at degenerating nerve terminals (both axonal anddendritic), and contain a core compound of amyloid protein fibers. Insummary, Alzheimer's disease is characterized by certainneuropathological features including intracellular neurofibrillarytangles, primarily composed of cytoskeletal proteins, and extracellularparenchymal and cerebrosvascular amyloid. Further, there are now methodsin the art of distinguishing between Alzheimer's patents, normal agedpeople, and people suffering from other neurodegenerative diseases, suchas Parkinson's, Huntington's chorea, Wernicke-Korsakoff or schizophreniafurther described for instance in U.S. Pat. No. 5,580,748 and U.S. Pat.No. 6,080,582.

While cellular changes leading to neuronal loss and the underlyingetiology of the disease remain under investigation the importance of APPmetabolism is well established. The two proteins most consistentlyidentified in the brains of patients with Alzheimer's disease to play arole in the physiology or pathophysiology of brain are β-amyloid andtau. (See Selkoe (2001) Physiological Reviews. 81:2). A discussion ofthe defects in β-amyloid protein metabolism and abnormal calciumhomeostasis and/or calcium activated kinases. (Etcheberrigaray et al.Alzheimer's Reports Vol. Nos. 3, 5 & 6 pp 305-312; Webb et al. (2000)British Journal of Pharmacology 130: 1433-52).

Alzheimer's disease (AD) is a brain disorder characterized by alteredprotein catabolism. Altered protein phosphorylation has been implicatedin the formation of the intracellular neurofibrillary tangles found inAlzheimer's disease. The processing of the amyloid precursor protein(APP) determines the production of fragments that later aggregateforming the amyloid deposits characteristic of Alzheimer's disease (AD),known as senile or AD plaques. A central feature of the pathology ofAlzheimer's disease is the deposition of amyloid protein within plaques.Thus, APP processing is an early and key pathophysiological event in AD.

Three alternative APP processing pathways have been identified. Thepreviously termed “normal” processing involves the participation of anenzyme that cleaves APP within the Aβ sequence at residue Lys16 (orbetween Lys16 and Leu17; APP770 nomenclature), resulting innon-amyloidogenic fragments: a large N-terminus ectodomain and a small 9kDa membrane bound fragment. This enzyme, yet to be fully identified, isknown as α-secretase. Two additional secretases participate in APPprocessing. One alternative pathway involves the cleavage of APP outsidethe Aβ domain, between Met671 and Asp672 (by β-secretase) and theparticipation of the endosomal-lysomal system. An additional cleavagesite occurs at the carboxyl-terminal end of the Aβ portion, within theplasma membrane after amino acid 39 of the Aβ peptide. The secretase (γ)action produces an extracellular amino acid terminal that contains theentire Aβ sequence and a cell-associated fragment of ˜6 kDa. Thus,processing by β and γ secretases generate potential amyloidogenicfragments since they contain the complete Aβ sequence. Several lines ofevidence have shown that all alternative pathways occur in a givensystem and that soluble Aβ may be a “normal product.” However, there isalso evidence that the amount of circulating Aβ in CSF and plasma iselevated in patients carrying the “Swedish” mutation. Moreover, culturedcells transfected with this mutation or the APP₇₁₇ mutation, secretelarger amounts of Aβ. More recently, carriers of other APP mutations andPS1 and PS2 mutations have been shown to secrete elevated amounts of aparticular form, long (42-43 amino acids) Aβ.

Therefore, although all alternative pathways may occur normally, animbalance favoring amyloidogenic processing occurs in familial andperhaps sporadic AD. These enhanced amyloidogenic pathways ultimatelylead to fibril and plaque formation in the brains of AD patients. Thus,intervention to favor the non-amyloidogenic, α-secretase pathwayeffectively shifts the balance of APP processing towards a presumablynon-pathogenic process that increases the relative amount of sAPPcompared with the potentially toxic Aβ peptides.

The PKC isoenzymes provides a critical, specific and rate limitingmolecular target through which a unique correlation of biochemical,biophysical, and behavioral efficacy can be demonstrated and applied tosubjects to improve cognitive ability.

Further with regard to normal and abnormal memory both K⁺ and Ca²⁺channels have been demonstrated to play key roles in memory storage andrecall. For instance, potassium channels have been found to changeduring memory storage. (Etcheberrigaray et al. (1992) Proc. Natl. Acad.Sci. 89: 7184; Sanchez-Andres et al. (1991) Journal of Neurobiology 65:796; Collin et al. (1988) Biophysics Journal 55: 955; Alkon et al.(1985) Behavioral and Neural Biology 44: 278; Alkon (1984) Science 226:1037). This observation, coupled with the almost universal symptom ofmemory loss in Alzheimer's patents, led to the investigation ofpotassium channel function as a possible site of Alzheimer's diseasepathology and the effect of PKC modulation on cognition.

3. Protein Kinase C and Alzheimer's Disease

PKC was identified as one of the largest gene families of non-receptorserine-threonine protein kinases. Since the discovery of PKC in theearly eighties by Nishizuka and coworkers (Kikkawa et al. (1982) J.Biol. Chem. 257: 13341), and its identification as a major receptor ofphorbol esters (Ashendel et al. (1983) Cancer Res., 43: 4333), amultitude of physiological signaling mechanisms have been ascribed tothis enzyme. The intense interest in PKC stems from its unique abilityto be activated in vitro by calcium and diacylglycerol (and its phorbolester mimetics), an effector whose formation is coupled to phospholipidturnover by the action of growth and differentiation factors.

The PKC gene family consists presently of 11 genes which are dividedinto four subgrounds: 1) classical PKCα, β₁, β₂ (β₁ and β₂ arealternatively spliced forms of the same gene) and γ, 2) novel PKCδ, ε, ηand θ, 3) atypical PKCζ, λ, η and ι and 4) PKCμ. PKCμ resembles thenovel PKC isoforms but differs by having a putative transmembrane domain(reviewed by Blohe et al. (1994) Cancer Metast. Rev. 13: 411; Ilug etal. (1993) Biochem J. 291: 329; Kikkawa et al. (1989) Ann. Rev. Biochem.58: 31). The α, β₁, β₂, and γ isoforms are Ca², phospholipid anddiacylglycerol-dependent and represent the classical isoforms of PKC,whereas the other isoforms are activated by phospholipid anddiacylglycerol but are not dependent on CA²⁺. All isoforms encompass 5variable (V1-V5) regions, and the α, β, γ isoforms contain four (C1-C4)structural domains which are highly conserved. All isoforms except PKCα,β and γ lack the C2 domain, and the λ, η and isoforms also lack nine oftwo cysteine-rich zinc finger domains in C1 to which diacylglycerolbinds. The C1 domain also contains the pseudo substrate sequence whichis highly conserved among all isoforms, and which serves an autoregulatory function by blocking the substrate-binding site to produce aninactive conformation of the enzyme (House et al., (1987) Science 238:1726).

Because of these structural features, diverse PKC isoforms are thoughtto have highly specialized roles in signal transduction in response tophysiological stimuli (Nishizuka (1989) Cancer 10: 1892), as well as inneoplastic transformation and differentiation (Glazer (1994) ProteinKinase C. J. F. Kuo, ed., Oxford U. Press (1994) at pages 171-198). Fora discussion of known PKC modulators see PCT/US97/08141, U.S. Pat. Nos.5,652,232; 6,043,270; 6,080,784; 5,891,906; 5,962,498; 5,955,501;5,891,870 and 5,962,504.

In view of the central role that PKC plays in signal transduction, PKChas proven to be an exciting target for the modulation of APPprocessing. It is well established that PKC plays a role in APPprocessing. Phorbol esters for instance have been shown to significantlyincrease the relative amount of non-amyloidogenic soluble APP (sAPP)secreted through PKC activation. Activation of PKC by phorbol ester doesnot appear to result in a direct phosphorylation of the APP molecule,however. Irrespective of the precise site of action, phorbol-induced PKCactivation results in an enhanced or favored α-secretase,non-amyloidogenic pathway. Therefore PKC activation is an attractiveapproach for influencing the production of non-deleterious sAPP and evenproducing beneficial sAPP and at the same time reduce the relativeamount of Aβ peptides. Phorbol esters, however, are not suitablecompounds for eventual drug development because of their tumor promotionactivity. (Ibarreta et al. (1999) NeuroReport Vol. 10, No. 5&6, pp1034-40).

The present inventors have also observed that activation of proteinkinase C favors the α-secretase processing of the Alzheimer's disease(AD) amyloid precursor protein (APP), resulting in the generation ofnon-amyloidogenic soluble APP (sAPP). Consequently, the relativesecretion of amyloidogenic A₁₋₄₀ and A₁₋₄₂₍₃₎ is reduced. This isparticularly relevant since fibroblasts and other cells expressing APPand presenilin AD mutations secrete increased amounts of total Aβ and/orincreased ratios of A₁₋₄₂₍₃₎/A₁₋₄₀. Interesting, PKC defects have beenfound in AD brain (α and β isoforms) and in fibroblasts (α-isoform) fromAD patients.

Studies have shown that other PKC activators (i.e. benzolactam) withimproved selectivity for the α, β and γ isoforms enhance sAPP secretionover basal levels. The sAPP secretion in benzolactam-treated AD cellswas also slightly higher compared to control benzolactam-treatedfibroblasts, which only showed significant increases of sAPP secretionafter treatment with 10 μM BL. It was further reported thatstaurosporine (a PKC inhibitor) eliminated the effects of benzolactam inboth control and AD fibroblasts while related compounds also cause a˜3-fold sAPP secretion in PC12 cells. The present inventors have foundthat the use of bryostatin as a PKC activators to favornon-amyloidogenic APP processing is of particular therapeutic valuesince it is non-tumor promoting and already in stage II clinical trials.

Alterations in PKC, as well alterations in calcium regulation andpotassium (K⁺) channels are included among alterations in fibroblasts inAlzheimer's disease (AD) patients. PKC activation has been shown torestore normal K⁺ channel function, as measured by TEA-induced [Ca²⁺]elevations. Further patch-clamp data substantiates the effect of PKCactivators on restoration of 113 psK⁺ channel activity. Thus PKCactivator-based restoration of K⁺ channels has been established as anapproach to the investigation of AD pathophysiology, and provides auseful model for AD therapeutics. (See, pending U.S. application Ser.No. 09/652,656, which is incorporated herein by reference in itsentirety.)

Of particular interest are macrocyclic lactones (i.e. bryostatin classand neristatin class) that act to stimulate PKC. Of the bryostatin classcompounds, bryostatin-1 has been shown to activate PKC and proven to bedevoid of tumor promotion activity. Bryostatin-1, as a PKC activator, isalso particularly useful since the dose response curve of bryostatin-1is biphasic. Additionally, bryostatin-1 demonstrates differentialregulation of PKC isozymes, including PKCα, PKCδ, and PKCε. Bryostatin-1has undergone toxicity and safety studies in animals and humans and isactively being investigated as an anti-cancer agent. Bryostatin-1's usein the studies has determined that the main adverse reaction in humansis myalgia, limiting the maximum dose to 40 mg/m². The present inventionhas utilized concentrations of 0.1 nM of bryostatin-1 to cause adramatic increase of sAPP secretion. Bryostatin-1 has been compared to avehicle alone and to another PKC activator, benzolactam (BL), used at aconcentration 10,000 times higher. Bryostatin is currently in clinicaltrials as an anti-cancer agent. The bryostatins are known to bind to theregulatory domain of PKC and to activate the enzyme. Bryostatin is anexample of isozyme-selective activators of PKC. Compounds in addition tobryostatins have been found to modulate PKC. (See, for example, WO97/43268; incorporated herein by reference in its entirety).

Macrocyclic lactones, and particularly bryostatin-1 is described in U.S.Pat. No. 4,560,774 (incorporated herein by reference in its entirety).Macrocyclic lactones and their derivatives are described elsewhere inthe art for instance in U.S. Pat. No. 6,187,568, U.S. Pat. No.6,043,270, U.S. Pat. No. 5,393,897, U.S. Pat. No. 5,072,004, U.S. Pat.No. 5,196,447, U.S. Pat. No. 4,833,257, and U.S. Pat. No. 4,611,066(each of which are incorporated herein by reference in theirentireties). The above patents describe various compounds and varioususes for macrocyclic lactones including their use as ananti-inflammatory or anti-tumor agent. Other discussions regardingbryostatin class compounds can be found in: Szallasi et al. (1994)Differential Regulation of Protein Kinase C Isozymes by Bryostatin 1 andPhorbol 12-Myristate 13-Acetate in NIH 3T3 Fibroblasts, Journal ofBiological Chemistry 269(3): 2118-24; Zhang et al. (1996) PreclinicalPharmacology of the Natural Product Anticancer Agent Bryostatin 1, anActivator of Protein Kinase C, Cancer Research 56: 802-808; Hennings etal. (1987) Bryostatin 1, an activator of protein kinase C, inhibitstumor promotion by phorbol esters in SENCAR mouse skin, Carcinogenesis8(9): 1343-46; Varterasian et al. (2000) Phase II Trial of Bryostatin 1in Patients with Relapse Low-Grade Non-Hodgkin's Lymphoma and ChronicLymphocytic Leukemia, Clinical Cancer Research 6: 825-28; and Mutter etal. (2000) Review Article: Chemistry and Clinical Biology of theBryostatins, Bioorganic & Medicinal Chemistry 8: 1841-1860 (each ofwhich is incorporated herein by reference in its entirety).

Myalgia is the primary side effect that limits the tolerable dose of aPKC activator. For example, in phase II clinical trials usingbryostatin-1, myalgia was reported in 10 to 87% of all treated patients.(Clamp et al. (2002) Anti-Cancer Drugs 13: 673-683). Doses of 20 μg/m²once per week for 3 weeks were well tolerated and were not associatedwith myalgia or other side effects. (Weitman et al. (1999) ClinicalCancer Research 5: 2344-2348). In another clinical study, 25 μg/m2 ofbryostatin-1 administered once per week for 8 weeks was the maximumtolerated dose. (Jayson et al. (1995) British J. of Cancer 72(2):461-468). Another study reported that 50 μg/m² (a 1 hour i.v. infusionadministered once every 2 weeks for a period of 6 weeks) was themaximum-tolerated dose. (Prendville et al. (1993) British J. of Cancer68(2): 418-424). The reported myalgia was cumulative with repeatedtreatments of bryostatin-1 and developed several days after initialinfusion. Id. The deleterious effect of myalgia on a patient's qualityof life was a contributory reason for the discontinuation ofbryostatin-1 treatment. Id. The etiology of bryostatin-induced myalgiais uncertain. Id.

The National Cancer Institute has established common toxicity criteriafor grading myalgia. Specifically, the criteria are divided into fivecategories or grades. Grade 0 is no myalgia. Grade 1 myalgia ischaracterized by mild, brief pain that does not require analgesic drugs.In Grade 1 myalgia, the patient is fully ambulatory. Grade 2 myalgia ischaracterized by moderate pain, wherein the pain or required analgesicsinterfere with some functions, but do not interfere with the activitiesof daily living. Grade 3 myalgia is associated with severe pain, whereinthe pain or necessary analgesics severely interfere with the activitiesof daily living. Grade 4 myalgia is disabling.

The compositions of the present invention increase the tolerable dose ofthe PKC activator administered to a patient and/or ameliorate the sideeffects associated with PKC activation by attenuating the activation ofPKC in peripheral tissues. Specifically, PKC inhibitors inhibit PKC inperipheral tissues or preferentially inhibit PKC in peripheral tissues.Vitamin E, for example, has been shown to normalizediacylglycerol-protein kinase C activation in the aorta of diabetic ratsand cultured rat smooth muscle cells exposed to elevated glucose levels.(Kunisaki et al. (1994) Diabetes 43(11): 1372-1377). In a double-blindtrial of vitamin E (2000 IU/day) treatment in patients suffering frommoderately advanced Alzheimer's Disease, it was found that vitamin Etreatment reduced mortality and morbidity, but did not enhance cognitiveabilities. (Burke et al. (1999) Post Graduate Medicine 106(5): 85-96).

Macrocyclic lactones, including the bryostatin class were originallyderived from Bigula neritina L. While multiple uses for macrocycliclactones, particularly the bryostatin class are known, the relationshipbetween macrocyclic lactones and cognition enhancement was previouslyunknown.

The examples of the compounds that may be used in the present inventioninclude macrocyclic lactones (i.e. bryostatin class and neristatin classcompounds). While specific embodiments of these compounds are describedin the examples and detailed description, it should be understood thatthe compounds disclosed in the references and derivatives thereof couldalso be used for the present compositions and methods.

As will also be appreciated by one of ordinary skill in the art,macrocyclic lactone compounds and their derivatives, particularly thebryostatin class, are amenable to combinatorial synthetic techniques andthus libraries of the compounds can be generated to optimizepharmacological parameters, including, but not limited to efficacy andsafety of the compositions. Additionally, these libraries can be assayedto determine those members that preferably modulate α-secretase and/orPKC.

Synthetic analogs of bryostatin are also contemplated by the presentinvention. Specifically, these analogues retain the orientation of theC1-, C19-, C26-oxygen recognition domain as determined by NMRspectroscopic comparison with bryostatin and various degrees ofPKC-binding affinity. The bryostatin analogues disclosed and describedin U.S. Pat. No. 6,624,189 (incorporated herein by reference in itsentirety) may also be used in the methods of the present invention.Specifically, the bryostatin analogues described by the genus of FormulaI of U.S. Pat. No. 6,624,189 (column 3, lines 35-66) and the species offormulas II-VII and 1998a and 1998b (column 8, lines 28-60) of U.S. Pat.No. 6,624,189 are PKC activators suitable for use in the methods of thepresent invention.

There still exists a need for the development of methods for thetreatment for improved overall cognition, either through a specificcharacteristic of cognitive ability or general cognition. There alsostill exists a need for the development of methods for the improvementof cognitive enhancement whether or not it is related to specificdisease state or cognitive disorder. The methods and compositions of thepresent invention fulfill these needs and will greatly improve theclinical treatment for Alzheimer's disease and other neurodegenerativediseases, as well as, provide for improved cognitive enhancement. Themethods and compositions also provide treatment and/or enhancement ofthe cognitive state through the modulation of α-secretase.

EXAMPLES Example 1 Behavioral Pharmacology

Bryostatin exposure—Specimens of Hermissenda Crassicornis weremaintained in artificial sea water (ASW) at 15° for three days inperforated 50-ml conical centrifuge tubes before starting experiments.Bryostatin, purified from the marine bryozoan Bugula neritina, wasdissolved in EtOH and diluted to its final concentration in ASW. Animalswere incubated with bryostatin in ASW for 4 hr, then rinsed with normalASW. For selected experiments lactacysteine (10 μM) or anisomycin wasadded to the ASW.

Bryostatin effects on Hermissenda behavior and biochemistry wereproduced by adding the drug to the bathing medium within an 8 cm long, 1cm diameter test tube housing each individual animal.

Example 2 Immunostaining Methods

Following experimental treatments and testing, animals were rapidlydecapitated, the central nervous systems (CNS) removed and then fixed in4% para-formaldehyde in 20 mM Tris-buffered (pH 8) natural seawater(NSW; 0.2 μm micropore-filtered). The CNSs were then embedded inpolyester wax (20), sectioned (6 μm) and immunostained using abiotinylated secondary antibody coupled to avidin-bound microperoxidase(ABC method, Vector), Aminoethylcarbazole (AEC) was used as thechromogen. The primary polyclonal antibody (designated 25U2) was raisedin rabbits from the full length calexcitin protein extracted from squidoptic lobes. Gray-scale intensity measures were done from digitalphotomicrographs on circumscribed cytoplasmic areas of theB-photoreceptors minus the same background area (non-stainingneuropile).

Example 3 Protein Kinase C Assay

Cells were homogenized by sonication (5 sec, 25 W) in 100 μl of 10 mMTris-HCl pH 7.4 buffer containing 1 mM EGTA, 1 mM PMSF, and 50 mM NaF.Homogenate was transferred to a polyallomer centrifuge tube and wascentrifuged at 100,000×g for 10 min at 4°. The supernatant was removedand immediately frozen on dry ice. The particulate fraction wasresuspended by sonication in 100 μl of the same buffer and stored at−80°. To measure PKC, 10 μl of cytosol or particulate fraction wasincubated for 15 min at 37° in the presence of 10 μM histones, 4.89 mMCaCI₂, 1.2 μg/μl phosphatidyl-L-serine, 0.18 μg/μl1.2-dioctanoyl-sn-glycerol, 10 mM MgCI₂, 20 mM HEPES (pH 7.4), 0-8 mMEDTA, 4 mM EGTA, 4% glycerol, 8 μg/ml aprotinin, 8 μg/ml leupeptin, and2 mM benzamidine. 0.5 μCi [γ³²P]ATP was added and ³²P-phosphoproteinformation was measured by adsorption onto phosphocellulose as describedpreviously (25). This assay was used with slight adjustments for eitherHermissenda nervous system homogenates or cultured mammalian neuronhomogenates

Example 4 Cell Culture

Rat hippocampal H19-7/IGF-IR cells (ATCC) were plated onto poly-L-lysinecoated plates and grown at 35° in DMEM/10% FCS for several days untilapprox. 50% coverage was obtained. The cells were then induced todifferentiate into a neuronal phenotype by replacing the medium with 5ml N2 medium containing 10 ng/ml basic fibroblast growth factor andgrown in T-25 flasks at 39° C. (26). Various concentrations ofbryostatin (0.01-1.0 nM) were then added in 10 μl aqueous solution.After a specified interval, the medium was removed and the cells werewashed with PBS, removed by gentle scraping, and collected bycentrifugation at 1000 rpm for 5 min.

Example 5 Behavioral Conditioning

Pavlovian conditioning of Hermissenda involves repeated pairings of aneutral stimulus, light, with an unconditioned stimulus, orbitalshaking. (See, Lederhendler et al. (24) and Epstein et al. (6)). Arotation/shaking stimulus excites the statocyst hair cells and therebyelicits an unconditioned response: a brisk contraction of the muscularundersurface called a foot, accompanied by adherence or “clinging” tothe surface that supports the foot. Before conditioning, light elicits aweakly positive phototaxis accompanied by lengthening of the foot. Aftersufficient light-rotation pairings, light no longer elicits phototaxis,but instead elicits a new response (24): the “clinging” and footshortening previously elicited only by the unconditional stimulus (FIG.1). Thus, the meaning of the unconditioned stimulus, rotation or orbitalshaking, has been transferred to the conditioned stimulus and ismanifested by a light-elicited foot contraction—a negative change offoot length. This conditioned response to light can last for weeks, isnot produced by randomized light and rotation, is stimulus-specific, andshares the other defining characteristics of mammalian PavlovianConditioning.

Example 6 Bryostatin-Induced Prolongation of Associative Memory

Pavlovian conditioning of Hermissenda has well-defined trainingparameters that produce progressively longer-lasting retention of thelearned conditioned response. Two training events (2 TE) of paired lightand orbital shaking (see “Methods”), for example, induce a learnedconditioned response (light-elicited foot contraction or shortening)that persists without drug treatment for approximately 7 minutes. Fourto six training events (4-6 TE) induce a conditioned response thatpersists up to several hours, but disappears approximately by 1 dayafter training. Nine TE produces long-term associative memory lastingmany days and often up to two weeks.

Animals were trained with sub-optimal regimes of 4- and 6-paired CS/UStraining events (TEs) with bryostatin (0.25 ng/ml) added during darkadaptation (10 min) prior to training and remaining for 4 hours, orwithout Bryo (NSW controls); 9-paired TEs and NSW served as the positivecontrols. All animals were tested with the CS alone at 4 h, then at 24-hintervals. Animals trained sub-optimally but treated with bryostatin alldemonstrated long-term retention (n=8-16 animals/condition/experiment;ANOVA, p<0.01).

Two TE plus bryostatin produced memory retention lasting hours (vs.minutes without bryostatin), 4 TEs plus bryostatin extended retentionbeyond 24 hours (FIG. 1), and 6 TE plus bryostatin produced retentionlasting 1 week or longer.

Without Bryostatin (NSW), random, and paired CS/US training events (TEs)did not generate LTM or elicit a CR when tested at 4 h. Bryostatin (0.25ng/ml in NSW) applied before 6-TE conditioning (during 10 min darkadaptation) and for 4 hours thereafter produced a positive CR (footcontraction; negative change in length), thus indicating LTM wasestablished. The antagonist, Ro-32 when applied pre-training (duringdark adaptation), blocked the effects of 6 TE plus bryostatin, i.e.animals lengthened (positive length change) with normal phototaxis(n=4-8 animals/condition/experiment; ANOVA differences, p<0.01).Randomized presentations of light and rotation, with or withoutbryostatin, produced no conditioned response (FIG. 2), i.e.,light-elicited foot-contraction. Thus, bryostatin during and immediatelyfollowing training prolonged memory retention with sub-optimal trainingtrials.

Example 7 Pre-Exposure to Bryostatin on Days Before Training EnhancesMemory Acquisition

Previous measurements (15, 17) have indicated that learning-induced PKCassociation with neuronal membranes (i.e., translocation) can besustained. Rabbit nictitating membrane conditioning, rat spatial mazelearning, maze learning, and rat olfactory discrimination learning haveall been found to be accompanied by PKC translocation that lasts fordays following training. Hermissenda conditioning was followed for atleast one day after training by PKC translocation that could belocalized in single, identifiable Type B cells (15).

As already described, exposure to bryostatin for 4 hours during andafter training enhances memory retention produced by 2 TE from 6-8minutes to several hours. However, a 4 hour exposure to bryostatin onthe day preceding training, as well as on the day of the 2 TE prolongedmemory retention for more than one day after training. Two successivedays of 4-h bryostatin exposure (0.25 ng/ml) of animals coupled with2-paired CS/US training events produced at least 6 days of long-termretention demonstrated by the CR (body length contraction) when testedwith the CS alone (n=16 animals/condition; ANOVA, p<0.01) (FIG. 3).

Animals given three successive days of 4-h bryostatin exposure (0.25ng/ml) followed one day later by 2-TEs, demonstrated long-term retention(LTR) measured over 96 h post-training. Non-exposed animals (same as inFIG. 3) did not demonstrate any behavioral modification (no CR to CStesting). Anisomycin (ANI) (1 μg/ml) administered immediately andremaining for four hours post-training to animals receiving thethree-day bryostatin treatments did not prevent long-term retention.Thus the requirement for protein synthesis necessary to generate LTRthat is usually blocked by ANI when added post-training was obviated bythe three-day bryostatin treatment (n=16 animals/condition; ANOVA,p<0.01). A third day of exposure to the 4 hour interval of bryostatincaused a similar enhanced retention of the Pavlovian conditionedresponse (FIG. 4). The preceding results support the view that twosuccessive intervals of exposure to bryostatin cause PKC activation andpossibly synthesis of proteins critical for long-term memory, with aminimum of concurrent and subsequent PKC downregulation. This view wasgiven further support by the observation that a more prolonged intervalof bryostatin exposure, i.e. for 8 to 20 hours, followed by 2 TE (FIG.5) was not sufficient itself to produce memory retention equivalent tothat which accompanied the two 4 hour exposures on successive precedingdays. In these experiments, the effects of 20 hr bryostatin (0.25 ng/ml)exposure on training was observed. With the sub-optimal 2-paired TEconditioning regime, retention was gone in 48 hours. Retention of4-paired TE conditioning with 20 h pre-exposure to bryostatin persisted(n=8 animals/condition; ANOVA at 48-h, p<0.01). Sufficiently prolongedbryostatin exposure (e.g., 8-12 hours) is known in other cell systems tocause prolonged PKC downregulation that may offset PKC activation andincrease PKC synthesis.

Similarly, sufficiently increased concentrations of bryostatinultimately blocked memory retention (FIG. 6) presumably also because ofPKC downregulation. Bryostatin concentrations <0.50 ng/ml augmentacquisition and memory retention with sub-optimal (4 TE) trainingconditions. Those concentrations had no demonstrable effects onretention performance with 9-paired TEs. However, with all trainingconditions tested, concentration ≧1.0 ng/ml inhibited acquisition andbehavioral retention (n=16 animals/condition), presumably via PKCdownregulation.

Example 8 Pre-Exposure to Bryostatin Obviates the Requirement forProtein Synthesis During Training

Animals received 2-paired training events (TEs) and then tested forretention after 4 h. Bryostatin (0.25 ng/ml) applied in NSW to animalsduring the 10-min pre-training dark adaptation period and 4 h thereafterdemonstrated retention of the behavioral conditioning (foot contraction(CR) and shortening in body length). NSW control animals and thosetreated with bryostatin pre-training followed by anisomycin (1.0 μg/ml)immediately post-training showed no CR with the foot lengthening innormal positive phototaxis (n=12 animals/condition/experiment, two-wayANOVA statistics, p<0.01). A single 4 hour exposure to bryostatintogether with 2 TE produced long-term memory retention lasting hoursthat was entirely eliminated when anisomycin was present along with thebryostatin (FIG. 7). Similar blocking effects of anisomycin were alsoobserved with 6 TE plus bryostatin. Repeated brief exposures tobryostatin, however, increase the net synthesis of PKC, calexcitin, andother memory proteins and thus eliminate the requirement for newsynthesis during and after Pavlovian conditioning—if PKC downregulationwere sufficiently minimized. Protein synthesis was blocked for 4 hourswith anisomycin immediately after 2 TE of animals that on each of 3preceding days had been first exposed to 4 hours of bryostatin. In thiscase, anisomycin-induced blockade of protein synthesis did not preventmemory retention that lasted many days (FIG. 4). By contrast, the same 4hour anisomycin treatment eliminated all memory retention produced by 9TE, a training regimen ordinarily followed by 1-2 weeks of memoryretention (27). Finally, if 2 TE were given one day after threesuccessive days of 4 hour exposures to bryostatin that was accompaniedeach time by anisomycin, long-term memory was eliminated.

Example 9 Pre-Exposure to Proteasome Inhibition Enhances BryostatinEffects on Memory

Another means of enhancing and prolonging de novo synthesis of PKC andother memory-related proteins is provided by blocking pathways involvedin protein degradation. One of these, the ubiquitin-proteasome pathway(28-30), is known to be a major route for degradation of the α-isozymeof PKC. Degradation of PKC-α has been previously shown to be largelyprevented by 20 μM-5 QμM of the proteasome inhibitor, Lactacysteine.

Animals were incubated simultaneously for 4 h with bryostatin (0.25ng/ml) and lactacysteine (10 μ/M), and then 24 hrs later wereconditioned with 2-paired CS/US training events (TEs). Animals weresubsequently tested with the CS alone at 4 h post-training and then at24-h intervals. Retention of the conditioned behavior was persistentwith the combined bryostatin/lactacysteine treatment; behavioralretention was lost by bryostatin-only-treated animals after 24 h.Lactacysteine-only treated animals showed no acquisition or retention ofbehavioral training (data not graphed). (n=28 animals, combinedbryostatin/lactacysteine; n=20, bryostatin alone; n=16, lactacysteinealone). Lactacysteine, in this case, transformed the short-term memoryproduced by the single bryostatin exposure (followed by 2 TE) tolong-term memory lasting many days (FIG. 8).

Example 10 Calexcitin-Immunostaining Due to PKC Activation

Recently we showed that an immunostaining label of calexcitin increasedwithin single identified Type B cells during acquisition and retentionof Hermissenda conditioning (20). Many previous findings have implicateda low molecular weight calcium and GTP-binding protein, calexcitin, as asubstrate for PKC isozymes during Hermissenda conditioning (19).Calexcitin, now completely sequenced in some animal species, and shownto have significant homology with similar proteins in other species(31), undergoes changes of phosphorylation during and after HermissendaPavlovian conditioning. It is also a high affinity substrate for thealpha-isozyme of PKC and a low affinity substrate for β and gamma (19).

Micrographs (A, B) depict representative tissue sections fromHermissenda eyes that were immunolabeled with the calexcitin polyclonalantibody, 25U2. Positive calexcitin immunostaining occurred in B-cellphotoreceptors (*B-Cell) of animals that experienced paired CS/UCSassociative conditioning with or without prior administration ofbryostatin (B). Random presentations of the two stimuli (trainingevents, TEs) did not produce behavioral modifications nor a rise incalexcitin above normal background levels (A); basement membrane andlens staining are artifact associated with using vertebrate polyclonalantibodies. Differences in staining intensities were measured andrecorded as gray-scale intensities (0-256; B-cell cytoplasm minus tissuebackground). Graph (C) displays intensity measures for Hermissendaconditioned with 9-random TEs (left bar) and animals treated with twoexposures on successive days to the PKC agonist, bryostatin (0.25ng/ml), and then associatively conditioned with 2-paired TEs. Activationof PKC from two exposures of bryostatin coupled with 2 TEs significantlyincreased calexcitin to levels associated with 9-paired TEs andconsolidated (long-term) memory (n=4-8 animals/condition/replicate;t-test comparison, p<0.01).

Calexcitin immunostaining is sufficiently sensitive to resolve boutonswithin synaptic fields of photic-vestibular neurites (D). Arrowsindicate arborization field between an interneuron (a), axon from acontralateral neuron (b), and terminal boutons of neurites from aputative photoreceptor (c). Scale bars=10 μm; CPG, cerebropleuralganglion (FIG. 9, 10).

This conditioning-induced calexcitin label increase represents anincrease in the actual amount of the protein since the immunostainingantibody reacts with both the phosphorylated and unphosphorylated formsof the protein. PKC, previously shown to translocate within the sameindividual Type B cells, apparently caused the conditioning-inducedincrease in the calexcitin label since the specific PKC-blocker, Ro-32,prevented both learning and learning-specific calexcitin increases inthe Type B cell (see above). Naïve and/or randomized control trainingprotocols produced a small fraction of the training-induced calexcitin(CE) immunostaining (FIG. 9).

Random training (4-TEs) without bryostatin yielded slightly higherintensity measures than background. Bryostatin administration increasedthe calexcitin levels for both training paradigms. With random training,when there was occasional overlap (pairing) of the CS and US, as was thecase here, it is not unexpected that some rise in CE might occur(increase of 2.0). However, calexcitin levels increased greater than4.3×with paired training (mean±SE, N=5 animals/treatment. 4 RTE=randomcontrol, 4 trials with random light and rotation; 6 PTE=paired trials, 6trials with paired light and rotation. 6 PTE-0 Bry vs. 6 PTE-0.25 Bry:p<0.001; 4 RTE-0.25 Bry vs. 6 PTE-0.25 Bry; p<0.001 (t-test). Whensub-optimal training events (4-6 TE) were used, the CE immunostaining(FIG. 10A) reached an intermediate level of elevation. These sub-optimalregimes were insufficient to produce memory retention lasting longerthan 24 hours. As described earlier, bryostatin administered duringtraining with 6 TE induced long-term memory retention (>1 week).Furthermore, bryostatin plus 6 TE induced CE immunostaining comparableto that observed after 9 TE.

Bryostatin in low doses (0.1-0.25 ng/ml) markedly enhanced memory after2, 4, or 6 training trials. Pavlovian conditioning with 6 TE producedmemory lasting many days with bryostatin, but lasting only hours withoutbryostatin. This memory enhancement was blocked by anisomycin or the PKCinhibitor, Ro-32. It is important to note that CE immunostaining wasgreatly reduced 24 hours after 9 TE even though the memory persisted formore than 1 week thereafter. More persistent CE immunostaining resulted,however, from repeated bryostatin exposures on days preceding minimaltraining (2 TE).

Bryostatin alone (without associative conditioning) administered for4-hr over each of 1, 2, and 3 days progressively increased the levels ofcalexcitin in the B-photoreceptors of Hermissenda when measured 24 hoursafter each of the periods of bryostatin exposures. Twenty-four hoursafter 1 bryostatin exposure for four hours, CE immunostaining was notelevated (FIG. 10B). Twenty-four hours after 2 bryostatin exposures, 1on each of two successive days showed greater residual CEimmunostaining. The calexcitin level after 3 bryostatin exposuresfollowed by just 2-paired training events (paired light and orbitalshaking) raised that level even higher with a significant concomitantlength in the number of retention days for the associativeconditioning-induced behavioral modification (n=16 animals/condition:ANOVA, p<0.01). With 2 TE on the subsequent day after these threeexposures, CE immunostaining 24 hours later approached the levelspreviously observed immediately following 9 TE (FIG. 10B). Thus, CEimmunostaining following these three days of 4 hour bryostatin exposurefollowed by minimal training (2 TE) showed a greater persistence thandid the training trials alone. This persistence of newly synthesizedcalexcitin is consistent with the biochemical observations indicatingenhanced protein synthesis induced by bryostatin.

Exposure to 4-hr of bryostatin on two consecutive days followed 24 hourslater by 2-training events (2 TE) are required to raise calexcitinlevels to the amount associated with consolidated long-term memory.Typically, 2-TEs with two bryostatin exposures produces retentionlasting more than one week (n=16 animals/condition; t-test, p<0.01).Priming with 4-hr exposures to bryostatin over 3 consecutive days willinduce calexcitin levels required for consolidated memory. Anisomycinadded immediately after the 2-paired training events did not reduce thiscalexcitin level and consolidated memory persists for many days (N=8animals/condition; t-test, p>0.05, ns). (FIGS. 11 A, B).

It is noteworthy that the Ro-32 inhibition of PKC immediately afterbryostatin plus training did not prevent long-term memory induction,while this inhibition during the training plus bryostatin did preventmemory consolidation. In contrast, anisomycin during training with andwithout bryostatin did not prevent long-term memory, while anisomycinafter training with and without bryostatin completely blocked memoryformation. Therefore, PKC activation during training is followed byprotein synthesis required for long-term memory. Thus, once PKCactivation is induced to sufficient levels, the required proteinsynthesis is an inevitable consequence. Consistently, bryostatin-inducedPKC activation on days prior to training is sufficient, with minimaltraining trials, to cause long-term memory. Furthermore, this latterlong-term memory does not require protein synthesis following thetraining (and PKC activation on preceding days). Again, prior PKCactivation was sufficient to produce that protein synthesis necessaryfor subsequent long-term memory formation. One of those proteins whosesynthesis is induced by bryostatin-induced PKC activation as well asconditioning trials is calexcitin—as demonstrated by the immunostaininglabeling. The other protein is PKC itself.

Example 11 Effect of Bryostatin on PKC Activity

Bryostatin is known to transiently activate PKC by increasing PKCassociation with the cellular membrane fraction. A variety ofassociative memory paradigms have also been demonstrated to causeincreased PKC association with neuronal membranes. We tested, therefore,the possibility that repeated exposures of Hermissenda to bryostatin(i.e., 4 hour exposures, exactly as with the training protocols) mightalso induce prolonged PKC activation.

Intact Hermissenda were exposed for 4 hour intervals to bryostatin (0.28nM) on successive days under conditions described (“BehavioralPharmacology”). Histone phosphorylation (See “Methods”) in isolatedcircumesophageal nervous systems was then measured in the cytosolfraction. PKC activity measured both 10 minutes and 24 hours after thesecond of two bryostatin exposures was significantly increased overbaseline levels (N=6, for each measurement). (FIG. 12, 13). Thus, thequantity of PKC in both fractions was apparently increased, but not theratio of the PKC in the membrane to that in the cytosolic fraction.These results demonstrate that the bryostatin pre-exposure causes aneffect on PKC somewhat different from learning itself. After an initialactivation (via translocation), this bryostatin effect is most likelydue to increased synthesis of PKC, consistent with the increased levelsof calexcitin induced by bryostatin, but not directly correlated withrepeated bryostatin exposure.

As in FIG. 12, 13 but with anisomycin (1.0 ng/ml) added together witheach bryostatin (0.25 ng/ml) exposure. Note that the anisomycin markedlyreduced the PKC activity in both the cytosolic and membrane fractionsfrom the Hermissenda circumesophageal nervous systems after exposure tobryostatin on three successive days (N=3, for each measurement, p<0.01)(FIG. 14).

To further examine biochemical consequences of repeated exposures tobryostatin, rat hippocampal neurons were studied after they had beenimmortalized by retroviral transduction of temperature sensitivetsA5CSV40 large T antigen (25). These differentiate to have a neuronalphenotype when induced by basic fibroblast growth factor in N2 medium(26) and express a normal complement of neuronal proteins, includingPKC.

Exposure of cultured hippocampal neurons to a single activating dose ofbryostatin (0.28 nM) for 30 minutes produced a brief translocation ofPKC from the cytosol to the particulate fraction (approx 60%) followedby a prolonged downregulation (FIG. 15). Both the initial PKC activationand subsequent downregulation have been previously described and wereconfirmed by measurement of PKC activity in membrane and cytosol.Exposing the cultured hippocampal neurons to one 30-minute period ofbryostatin, followed by a second 30-minute exposure, at intervalsranging from 30 minutes to 8 hours, caused the membrane-bound PKC torebound more quickly. Thus, a second exposure after a 2- to 4-hour delayeliminated the significant downregulation that a single bryostatinexposure produced (FIG. 16). In the cytoplasmic fraction, no significantalteration of PKC activity was detected within the first 4 hours afterbryostatin exposure. In contrast, if cells were exposed to bryostatintwice within a 2-hour period, there was a significant reduction of PKCactivity in response to the second exposure. However, if the secondexposure was delayed until 4 hours after the first, activity wasincreased above baseline, to a degree that was significantly greatercompared with a second exposure delivered after 2 hours or less (FIG.16).

These results are consistent with the interpretation that the initialbryostatin activation of PKC followed by downregulation (28-30) leads toincreased synthesis (via de novo protein synthesis) of PKC isozymes (aswell as calexcitin, described earlier). In fact, we found here that asingle 30-minute exposure to 0.28 nM bryostatin increased overallprotein synthesis (FIG. 17), measured by incorporation of ³⁵-SMethionine in the last ½ hour before collecting the neurons, by 20%within 24 h, increasing to 60% by 79 hours after the bryostatinexposure. This prolonged and profound increase of protein synthesisinduced by bryostatin was partially blocked when the PKC inhibitor Ro-32was also present (FIG. 17).

Abundant observations indicate that sufficient bryostatin-induced PKCactivation leads, inevitably, to progressive PKC inactivation andsubsequent downregulation. Sufficient doses of bryostatin (greater than1.0 ng/ml) actually inhibited Pavlovian conditioning. This was mostlikely due to PKC downregulation that characterized the behavioralresults with high bryostatin concentrations. PKC activation induced bybryostatin has been shown to be downregulated by two distinct pathways.One that is also induced by phorbol ester involves ubiquitination andsubsequent proteolytic degradation through the proteasome pathway. Thesecond mechanism of downregulation, not induced by phorbol ester,involves movement through caveolar compartments and degradation mediatedby phosphatase PP1 and PP2A. With sufficient concentrations and/ordurations of PKC activators, the PKC degrading pathways create a deficitof PKC that stimulates de novo synthesis of PKC, PKC synthesis cannotcompensate for inactivation and down regulation, thereby causingdepletion of available PKC of 95% or more.

1. A method comprising the step of contacting a PKC activator with aprotein kinase C (PKC) to stimulate the synthesis of proteins sufficientto consolidate long term memory.
 2. The method of claim 1, wherein saidPKC activator is a macrocyclic lactone.
 3. The method of claim 1,wherein the PKC activator is a benzolactam.
 4. The method of claim 1,wherein the PKC activator is a pyrrolidinone.
 5. The method of claim 2,wherein the macrocyclic lactone is a bryostatin.
 6. The method of claim5, wherein the bryostatin is bryostatin-1, -2, -3, -4, -5, -6, -7, -8,-9, -10, -11, -12, -13, -14, -15, -16, -17, or -18.
 7. The method ofclaim 5, wherein the bryostatin is bryostatin-1.
 8. The method of claim2, wherein the macrocyclic lactone is a neristatin.
 9. The compositionof claim 8, wherein the neristatin is neristatin-1.
 10. The method ofclaim 1, wherein said contact activates PKC.
 11. The method of claim 1,wherein said contact increases the amount of PKC.
 12. The method ofclaim 1, wherein said contact increases the synthesis of PKC.
 13. Themethod of claim 1, wherein said contact increases the amount ofcalexcitin.
 14. The method of claim 1, wherein said contact does notresult in substantial subsequent downregulation of PKC.
 15. The methodof claim 1, wherein the contacting of the PKC activator with the PKC isrepeated.
 16. The method of claim 15, wherein the contacting of the PKCactivator with the PKC is repeated at regular intervals.
 17. The methodof claim 16, wherein the interval is between one week to one month, oneday and one week, or less than one hour and 24 hours.
 18. The method ofclaim 17, wherein the interval is between one week and one month. 19.The method of claim 17, wherein the interval is between one day and oneweek.
 20. The method of claim 17, wherein the interval is between lessthan one hour and 24 hours.
 21. The method of claim 1, wherein thecontacting of the PKC activator with the PKC is maintained for a fixedduration.
 22. The method of claim 21, wherein the fixed duration is lessthan 24 hours.
 23. The method of claim 21, wherein the fixed duration isless than 12 hours.
 24. The method of claim 21, wherein the fixedduration is less than 6 hours.
 25. The method of claim 21, wherein thefixed duration is less than 4 hours.
 26. The method of claim 21, whereinthe fixed duration is less than 2 hours.
 27. The method of claim 21,wherein the fixed duration is between about 2 and about 6 hours.
 28. Themethod of claim 21, wherein the fixed duration is about 4 hours.
 29. Themethod of claim 21, wherein said duration of said contact is betweenabout 1 and about 12 hours.
 30. The method of claim 15, wherein saidcontact is repeated for a period greater than one day.
 31. The method ofclaim 15, wherein said contact is repeated for a period between one dayand one month.
 32. The method of claim 15, wherein said contact isrepeated for a period between one day and one week.
 33. The method ofclaim 15, wherein said contact is repeated for a period between one weekand one month.
 34. The method of claim 15, wherein said contact isrepeated for a period between one month and six months.
 35. The methodof claim 15, wherein said contact is repeated for a period of one month.36. The method of claim 15, wherein said contact is repeated for aperiod greater than one month.
 37. A method comprising the step ofcontacting a PKC activator with a protein kinase C (PKC) to downregulatePKC.
 38. The method of claim 37, wherein said PKC activator is amacrocyclic lactone.
 39. The method of claim 37, wherein the PKCactivator is a benzolactam.
 40. The method of claim 37, wherein the PKCactivator is a pyrrolidinone.
 41. The method of claim 38, wherein themacrocyclic lactone is a bryostatin.
 42. The method of claim 41, whereinthe bryostatin is bryostatin-1, -2, -3, -4, -5, -6, -7, -8, -9, -10,-11, -12, -13, -14, -15, -16, -17, or -18.
 43. The method of claim 42,wherein the bryostatin is bryostatin-1.
 44. The method of claim 38,wherein the macrocyclic lactone is a neristatin.
 45. The composition ofclaim 38, wherein the neristatin is neristatin-1.
 46. The method ofclaim 37, wherein said contact produces downregulation of PKC.
 47. Themethod of claim 46, wherein said contact produces substantialdownregulation of PKC.
 48. The method of claim 37, wherein said contactdoes not stimulate the synthesis of PKC.
 49. The method of claim 48,wherein said contact does not substantially stimulate the synthesis ofPKC.
 50. The method of claim 37, wherein said contact decreases theamount of PKC.
 51. The method of claim 50, wherein said contactsubstantially decreases the amount of PKC.
 52. The method of claim 37,wherein said contact does not stimulate the synthesis of calexcitin. 53.The method of claim 50, wherein said contact does not stimulate thesynthesis of calexitin.
 54. The method of claim 37, wherein thecontacting of the PKC activator with the PKC is for a sustained period.55. The method of claim 54, wherein the sustained period is between lessthan one hour and 24 hours.
 56. The method of claim 54, wherein thesustained period is between one day and one week.
 57. The method ofclaim 54, wherein the sustained period is between one week and onemonth.
 58. The method of claim 54, wherein the sustained period isbetween less than one hour and 12 hours.
 59. The method of claim 54,wherein the sustained period is between less than one hour and 8 hours.60. The method of claim 54, wherein the sustained period is between lessthan one hour and 4 hours.
 61. The method of claim 54, wherein thesustained period is about 4 hours.
 62. The method of claim 37, whereinsaid contact produces sustained downregulation of PKC.
 63. The method ofclaim 1, further comprising the step of inhibiting degradation ofprotein kinase C (PKC).
 64. The method of claim 63, wherein saiddegradation is through ubiquitination.
 65. The method of claim 64,wherein said degradation is inhibited by lactacysteine.
 66. The methodof claim 1, wherein the PKC is human.
 67. The method of claim 1, whereinthe PKC activator is provided in the form of a pharmaceuticalcomposition comprising the PKC activator and a pharmaceuticallyacceptable carrier.
 68. The method of claim 67, wherein thepharmaceutical composition further comprises a PKC inhibitor.
 69. Themethod of claim 68, wherein the PKC inhibitor inhibits PKC in peripheraltissues.
 70. The method of claim 68, wherein the PKC inhibitorselectively inhibits PKC in peripheral tissues.
 71. The method of claim68, wherein the PKC inhibitor is a compound that reduces myalgiaassociated with the administration of a PKC to a subjects.
 72. Themethod of claim 68, wherein the PKC inhibitor is a compound thatincreases the tolerable dose of a PKC activator.
 73. The method of claim68, wherein the PKC inhibitor is vitamin E, vitamin E analogs, vitamin Esalts, calphostin C, thiazolidinediones, ruboxistaurin or combinationsthereof.